The Flaviviridae family includes three genera: Flavivirus, having as main representatives the virus of the yellow fever, the virus of dengue, the virus of the Japanese encephalite; the genera Hepacivirus (virus of hepatite C) and the genera of Pestivirus (virus of diarrhea bovine). Even though they belong to different genera, with distinct biological properties and without crossed serological reactivity, the virus of the 3 types share a great similarity in the viral morphology, in the genomic organization and in the replication strategy (Rice, C. M. 1996. Flaviviridae: the viruses and their replication, Third ed, vol. 1. Lippincott-Raven, Philadelphia, Pa.).
The virus of the yellow fever is the prototype of the genera Flavivirus from the family Flaviviridae, which includes about 70 virus. The flavivirus are small (40-60 nm), spherical, enclosed, with RNA genome of single strain, with the majority of these arbovirus called as such due to their transmission by arthropod-born viruses (“arthropod-borne viruses”), as mosquitoes or ticks, causing important diseases on man and animals.
FIG. 1 presents the genomic organization of the Flavivirus (Chambers, T. J., C. S. Hahn, R. Galler, and C. M. Rice. 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649-88). The genome is represented on the top part, with the indication of the 5′ and 3′ non translated sequences and the open reading phase of 10.862 nucleotides. On this reading phase, 5′→3′ direction, the three structural proteins (C, prM and E) and the seven genes to the non structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B e NS5) are codified. The arrows indicated the proteolitic clivage sites performed by the viral protease (NS2B/NS3); and the lozenges, the cleavages by the cellular signalase (occurs inside the endoplasmatic reticule). The asterisks indicate the glycosilation sites linked to asparagines.
The yellow fever virus (FIG. 1) has a genome constituted by one single RNA molecule with 10.862 nucleotides (nt), one CAP structure at the 5′ edge (m7GpppG, to be recognized by the ribosomes), 5′ region non translated short (118 nt) and a 3′ edge not poliadenilated (511 nt). Such data were obtained from the first nucleotide sequencing of flavivirus genome—the vaccine virus vacinal 17D-204 (Rice, C. M., E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss. 1985. Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science 229:726-33).
In the cytoplasm of the host cell, the viral RNA is used as a shape to the synthesis of the negative complementary strain, which, by its turn, will be the shape to the synthesis of more positive strains to be used in the set up of new viral particles. The replication is a semi conservative process and involves replicative intermediates, as well as replicative ways. The formation of viral particles occurs through the relationship of the viral nucleocapsid, with the envelope protein anchored on the membrane of the cellular Endoplasmatic Reticule (RER). The set up of viral particles occurs in very close association with the RER. The viral particles are carried through vesicles and, from that point, released by the exocytose through the Golgy system.
The RNA is also the viral messenger and the transduction of infected cells results in the synthesis of a polyprotein forerunner of 3.411 aminoacids, which, when proteolytically processed, create the 10 viral polypeptides. From the 5′ edge, the order of genes is C; prM/M; E; NS1; NS2A; NS2B; NS3; NS4A; NS4B and NS5. The three first genes codify the structural viral proteins, that means, the ones which form the virus together with the encapsid RNA molecule, being denominated as capsid (C, 12-14 kDa), membrane (M of 8 kDa, and its forerunner prM of 18-22 kDa) and envelope (E, 52-54 kDa). These three genes are transcoded in the first quarter of the genome. The remaining genome codifies the non structural proteins (NS), numbered from 1 to 5 (NS1 a NS5), in accordance with the order of synthesis (Rice, C. M., E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss. 1985. Nucleotide sequence of yellow fever virus: implications for flavivirus gene expression and evolution. Science 229:726-33).
Among the different. Flavivirus, three great non structural proteins have very well conserved sequences: NS1 (38-41 kDa), NS3 (68-70 kDa) and NS5 (100-103 kDa).
The first one (NS1) has an important role in the replication of the negative strand of RNA (Lindenbach, B. D., and C. M. Rice. 1999. Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function. J Virol 73:4611-21; Lindenbach, B. D., and C. M. Rice. 1997. trans-Complementation of yellow fever virus NS1 reveals a role in early RNA replication. J Virol 71:9608-17; Muylaert, I. R., T. J. Chambers, R. Galler, and C. M. Rice. 1996. Mutagenesis of the N-linked glycosylation sites of the yellow fever virus NS1 protein: effects on virus replication and mouse neurovirulence. Virology 222:159-68; Muylaert, I. R., R. Galler, and C. M. Rice. 1997. Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature-sensitive mutation which blocks RNA accumulation. J Virol 71:291-8). Released extracellularly as hexameric structure, may be located in the cellular surface. Antibodies against NS1 do not neutralize the viral infectivity, but exert protective immunity through mediation of the complement lyzing infected cells (Rice, C. M. 1996. Flaviviridae: the viruses and their replication, Third ed, vol. 1. Lippincott-Raven, Philadelphia, Pa.).
The second one, NS3, make up three distinct enzymatic activities: (1) protease, being responsible for the proteolytic process of the viral polyprotein in sites where the cellular protease does not act (Lee, E., C. E. Stocks, S. M. Amberg, C. M. Rice, and M. Lobigs. 2000. Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production. J Virol 74:24-32; Stocks, C. E., and M. Lobigs. 1995. Posttranslational signal peptidase cleavage at the flavivirus C-prM junction in vitro. J Virol 69:8123-6; Yamshchikov, V. F., and R. W. Compans. 1995. Formation of the flavivirus envelope: role of the viral NS2B-NS3 protease. J Virol 69:1995-2003; Yamshchikov, V. F., D. W. Trent, and R. W. Compans. 1997. Upregulation of signalase processing and induction of prM-E secretion by the flavivirus NS2B-NS3 protease: roles of protease components. Virol 71:4364-71); (2) helicase and (3) nucleotide-trifosfatase (Gorbalenya, A. E., E. V. Koonin, A. P. Donchenko, and V. M. Blinov. 1989. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res 17:4713-30; Wengler, G., and G. Wengler. 1993. The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity. Virology 197:265-73; Wu, J., A. K. Sera, R. J. Kuhn, and J. L. Smith. 2005. Structure of the Flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing. J Virol 79:10268-77). The two last ones give to this protein an important role also in the replication of the viral RNA.
The third one, NS5, is the greatest and most conserved viral protein, making up the viral RNA polimerase, since its sequence contains several structural elements characteristic of RNA polymerases (Chambers, T. J., C. S. Hahn, R. Geller, and C. M. Rice. 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649-88) and still exhibits RNA polimerase activity, dependent of RNA (Steffens, S., H. J. Thiel, and S. E. Behrens. 1999. The RNA-dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro. J Gen Virol 80 (Pt 10):2583-90).
The four small proteins NS2A, NS2B, NS4A and NS4B are not enough conserved in its aminoacid sequence, but not in its patterns of multiple hydrophobic parts. These small proteins were related, up to the moment, to some processes of viral propagation: NS2A seems to be necessary to the correct processing of NS1 (Falgout, B., R. Chanock, and C. J. Lai. 1989. Proper processing of dengue virus nonstructural glycoprotein NS1 requires the N-terminal hydrophobic signal sequence and the downstream nonstructural protein NS2a. J Virol 63:1852-60) and to the set up of the viral particle together with NS3 (Kummerer, B. M., and C. M. Rice. 2002. Mutations in the yellow fever virus nonstructural protein NS2A selectively block production of infectious particles. J Virol 76:4773-84); NS2B is associated with NS3, acting as a complex proteolitic viral cofactor (Chambers, T. J., A. Nestorowicz, S. M. Amberg, and C. M. Rice. 1993. Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J Virol 67:6797-807; Falgout, B., M. Pethel, {grave over (Y)}. M. Zhang, and C. J. Lai. 1991. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. J Virol 65:2467-75; Jan, L. R., C. S. Yang, D. W. Trent, B. Falgout, and C. J. Lai. 1995. Processing of non-structural Japanese encephalitis virus proteins: NS2B-NS3 complex and heterologous proteases. J Gen Virol 76 (Pt 3):573-80); NS4A would interact with NS1, allowing its integration in the citoplasmatic process of RNA replication (Lindenbach, B. D., and C. M. Rice. 1999. Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function. J Virol 73:4611-21). Considering that the synthesis of the viral RNA occurs in the cellular cytoplasm in association with membranes of RER, it is assumed that these viral hydrophobic viral proteins would be immersed in membranes and, through interactions with NS3 and NS5, they would be participating with them in complex viral replicatives.
Structural elements present in the non translated 5′ and 3′ edges (NTR) are also important in the replication and wrapping of the viral RNA (Chambers, T. J., C. S. Hahn, R. Galler, and C. M. Rice. 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649-88; Cologna, R., and R. Rico-Hesse. 2003. American genotype structures decrease dengue virus output from human monocytes and dendritic cells. J Virol 77:3929-38; Elghonemy, S., W. G. Davis, and M. A. Brinton. 2005. The majority of the nucleotides in the top loop of the genomic 3′ terminal stem loop structure are cis-acting in a West Nile virus infectious clone. Virology 331:238-46; Hanley, K. A., L. R. Manlucu, G. G. Manipon, C. T. Hanson, S. S. Whitehead, B. R. Murphy, and J. E. Blaney, Jr. 2004. Introduction of mutations into the non-structural genes or 3′ untranslated region of an attenuated dengue virus type 4 vaccine candidate further decreases replication in rhesus monkeys while retaining protective immunity. Vaccine 22:3440-8; Khromykh, A. A., H. Meka, K. J. Guyatt, and E. G. Westaway. 2001. Essential role of cyclization sequences in flavivirus RNA replication. J Virol 75:6719-28; Thurner, C., C. Witwer, I. L. Hofacker, and P. F. Stadler. 2004. Conserved RNA secondary structures in Flaviviridae genomes. Gen Virol 85:1113-24; Tilgner, M., T. S. Deas, and P. Y. Shi. 2005. The flavivirus-conserved penta-nucleotide in the 3′ stem-loop of the West Nile virus genome requires a specific sequence and structure for RNA synthesis, but not for viral translation. Virology 331:375-86; Tilgner, M., and P. Y. Shi. 2004. Structure and function of the 3′terminal six nucleotides of the west nile virus genome in viral replication. J Virol 78:8159-71; Yu, L., and L. Markoff. 2005. The topology of bulges in the long stem of the flavivirus 3′ stem-loop is a major determinant of RNA replication competence. J Virol 79:2309-24).
The protein C of the capsid interacts with the viral RNA, forming the viral nucleocapsid (Chambers, T. J., C. S. Hahn, R. Galler, and C. M. Rice. 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649-88). The protein prM is a glicosilated forerunner of the membrane protein. It is present on the surface of immature viral particles, with the cleavage by cellular proteases furina type at the level of the Golgy complex, before the release of viral particles, in such way that the mature virus contains the protein M. The role of the prM is to stabilize the protein E, avoiding the premature show off of the fusion peptide to the reduced pH found in the exocite via (Heinz, F. X., and S. L. Allison. 2003. Flavivirus structure and membrane fusion. Adv Virus Res 59:63-97). The retention of prM protein may affect the conformation and antigenicity of the protein E and reduce the infectivity, inhibiting the acid-dependent fusion.
On FIG. 2, the immature (intracellular form) and mature (extracellular form) viral particles of the Flavivirus are represented. The capsid of the virus has an icosahedra symmetry, but the shape is not necessarily the one presented on the Figure, which also shows the genome of the virus associated with the internal side of the capsid. Here are represented the envelope proteins (E) and its dimeric form, the protein of the membrane (M) and its forerunner (prM), which is still present in the envelope in an extracellular shape. Oppositely to the extracellular particles, the intracellular particles are not infective (Chambers, T. J., C. S. Hahn, R. Galler, and C. M. Rice. 1990. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649-88).
The protein E is the main component of the viral envelope. It promotes the linkage to glicoproteic receptors on the cellular surface and the internalization by dependent fusion of pH, processes that trigger a viral infection. This protein has multiple determinant antigens and it is the main target to the immune-protective response of the vertebrate host. Therefore, it plays a key role in the cellular infections, in the viral tropism, in virulence and in the immunity.
The discovery of the three-dimensional atomic structure of the protein E of the mature viral particle of flavivirus TBE (tick-borne encephalitis virus), reveals that this protein exists as a homodimers, about 110 kDa, with three defined spheres, anchored by the hydrophobic carboxylic edge on the envelope surface (Rey, F. A., F. X. Heinz, C. Mandl, C. Kunz, and S. C. Harrison. 1995. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 375:291-8). This model has been seen applied to all Flavivirus, contributing mainly to the detection of antigen tracers and the study of mutations linked to the increase or decrease of virulence (Arroyo, J., F. Guirakhoo, S. Fenner, Z. X. Zhang, T. P. Monath, and T. J. Chambers. 2001. Molecular basis for attenuation of neurovirulence of a yellow fever Virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-J E). J Virol 75:934-42; Guirakhoo, F., Z. Zhang, G. Myers, B. W. Johnson, K. Pugachev, R. Nichols, N. Brown, I. Levenbook, K. Draper, S. Cyrek, J. Lang, C. Fournier, B. Barrere, S. Delagrave, and T. P. Monath. 2004. A single amino acid substitution in the envelope protein of chimeric yellow fever-dengue 1 vaccine virus reduces neurovirulence for suckling mice and viremia/viscerotropism for monkeys. J Virol 78:9998-10008; Halstead, S. B., F. X. Heinz, A. D. Barrett, and J. T. Roehrig. 2005. Dengue virus: molecular basis of cell entry and pathogenesis, 25-27 Jun. 2003, Vienna, Austria. Vaccine 23:849-56; Hurrelbrink, R. J., and P. C. McMinn. 2003. Molecular determinants of virulence: the structural and functional basis for flavivirus attenuation. Adv Virus Res 60:1-42; Kolaskar, A. S., and U. Kulkarni-Kale. 1999. Prediction of three-dimensional structure and mapping of conformational epitopes of envelope glycoprotein of Japanese encephalitis virus. Virology 261:31-42; Lee, E., R. A. Hall, and M. Lobigs. 2004. Common E protein determinants for attenuation of glycosaminoglycan-binding variants of Japanese encephalitis and West Nile viruses. J Virol 78:8271-80; Lee, E., and M. Lobigs. 2000. Substitutions at the putative receptor-binding site of an encephalitic flavivirus alter virulence and host cell tropism and reveal a role for glycosaminoglycans in entry. J Virol 74:8867-75; Lee, E., C. E. Stocks, S. M. Amberg, C. M. Rice, and M. Lobigs. 2000. Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production. J Virol 74:24-32; Mandl, C. W., S. L. Allison, H. Holzmann, T. Meixner, and F. X. Heinz. 2000. Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site. J Virol 74:9601-9; Nickells, M., and T. J. Chambers. 2003. Neuroadapted yellow fever virus 17D: determinants in the envelope protein govern neuroinvasiveness for SCID mice. J Virol 77:12232-42; Ryman, K. D., H. Xie, T. N. Ledger, G. A. Campbell, and A. D. Barrett. 1997. Antigenic variants of yellow fever virus with an altered neurovirulence phenotype in mice. Virology 230:376-80; Shirato, K., H. Miyoshi, A. Goto, Y. Ako, T. Ueki, H. Kariwa, and I. Takashima. 2004. Viral envelope protein glycosylation is a molecular determinant of the neuroinvasiveness of the New York strain of West Nile virus. J Gen Virol 85:3637-45).
The bonding of protein E to cell receptors leads to the formation of de endocitic vesicles, covered by clatrine. After the internalization by endocitose mediated by receptor, the virus are released in the cytoplasm through conformation changes, induced by acidic pH which takes the peptide of fusion to be exposed after the trimerization of protein E (Bonaldo, M. C., R. C. Garratt, R. S. Marchevsky, E. S. Coutinho, A. V. Jabor, L. F. Almeida, A. M. Yamamura, A. S. Duarte, P. J. Oliveira, J. O. Lizeu, L. A. Camacho, M. S. Freire, and R. Galler. 2005. Attenuation of recombinant yellow fever 17D viruses expressing foreign protein epitopes at the surface. J Virol 79:8602-13; Bressanelli, S., K. Stiasny, S. L. Allison, E. A. Stura, S. Duquerroy, J. Lescar, F. X. Heinz, and F. A. Rey. 2004. Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation. Embo J 23:728-38; Heinz, F. X., and S. L. Allison. 2003. Flavivirus structure and membrane fusion. Adv Virus Res 59:63-97; Stiasny, K., S. Bressanelli, J. Lepault, F. A. Rey, and F. X. Heinz. 2004. Characterization of a membrane-associated trimeric low-pH-induced Form of the class II viral fusion protein E from tick-borne encephalitis virus and its crystallization. J Virol 78:3178-83).
In 1927, the virus which causes the yellow fever was isolated in the Rhesus (Macaca mulatta), through the straight inoculation of blood from an African patient named Asibi (Stokes A, B. J., Hudson N P. 1928, The transmission of yellow fever to Macacus rhesus. Rev Med. Virol. 11:141-148). After the set up of a pattern of an animal model sensitive to the virus, new perspectives showed up and the viral propagation and the clinical evaluation became possible. The Asibi virus, the original sample, is one of the most virulent among the yellow fever virus ever studied. When inoculated in monkeys, through subcutaneous via, in 4 to 7 days it caused death in 95% of the animals, and high rates of viremia are detected in the blood of theses infected animals.
The serial passage of Asibi cepa, in different types of cultivation, as described priorly, lead to the production of the parental 17D cepa, in the passage 180, to 17DD in the passage 195, and to 17D-204 cepa in the passage 204. The 17DD cepa was cultivated afterwards until the passage 243 and suffered 43 extra passages in chicken embryo (passage 286). The 17D-204 cepa, by its turn, created by cultivation, to Colombia 88 cepa, that by its turn, originated the different seed shares used in France (I. Pasteur, passage 235) and in the United States (Connaught, passage 234). The 17D-204 and 17DD virus are the two sub cepas of the 17D cepas used actually to produce vaccines in the world, which accumulated the genotype and phenotype differences due to the independent serial passages (Galler, R., P. R. Post, C. N. Santos, and Ferreira, II. 1998. Genetic variability among yellow fever virus 17D substrains. Vaccine 16:1024-8; Marchevsky, R. S., M. S. Freire, E. S. Coutinho, and R. Galler. 2003. Neurovirulence of yellow fever 17DD vaccine virus to rhesus monkeys. Virology 316:55-63; Post, P. R., R. de Carvalho, M. da Silva Freire, and R. Galler. 2001. The early use of yellow fever virus strain 17D for vaccine production in Brazil—a review. Mem Inst Oswaldo Cruz 96:849-57). However, both are equally immunogenic and safe for human vaccine (Camacho, L. A., S. G. Aguiar, M. D. Freire, M. D. Leal, J. P. Nascimento, T. Iguchi, J. A. Lozana, and R. H. Farias. 2005. Reactogenicity of yellow fever vaccines in a randomized, placebo-controlled trial. Rev Saude Publica 39:413-420; Camacho, L. A., S. Freire Mda, L. Leal Mda, S. G. Aguiar, J. P. Nascimento., T. Iguchi, A. Lozana Jde, and R. H. Farias. 2004. Immunogenicity of WHO-17D and Brazilian 17DD yellow fever vaccines: a randomized trial. Rev Saude Publica 38:671-8).
The attenuated alive virus vaccine of the yellow fever (FA) 17D strain, constitutes one of the best and safer vaccines nowadays, having a well established methodology of production and a serious quality control, including the monkey neurovirulence test. Besides, it promotes lifetime immunity (Monath, T. 2003. Yellow Fever Vaccine, 4th ed. W.B. Saunders Company, USA) and it is capable of inducing both cellular immune and humoral responses (Co, M. D., M. Terajima, J. Cruz, F. A. Ennis, and A. L. Rothman. 2002. Human cytotoxic T lymphocyte responses to live attenuated 17D yellow fever vaccine: identification of HLA-B35-restricted CTL epitopes on nonstructural proteins NS1, NS2b, NS3, and the structural protein E. Virology 293:151-63); in addition to being low cost and one single dose. Its use was estimated in 400 million doses.
Due to this, its characteristics make it appropriate for the development of 17D virus as a vaccine expression vector of the heterolog antigens.
But, for the development of the flavivirus, expressing heterolog antigens, it is necessary to:                (a) the sketch of strategies that allow the introduction of heterolog antigens, without compromise of the structure and replication of the virus;        (b) ensure that the construction of the cDNA (and the RNA transcripts) generate a non-pathogenic virus and moreover that the foreign sequence stays integrated in the viral genome; and        (c) guarantee that the FA recombinant virus, besides being attenuated, keeps the immunologic properties, expressing the heterolog antigens, inserted in a way that it induces the appropriated immune response. It is also important that the replication capacity in certified cells for production of vaccines is maintained.        
The development of the recombinant DNA technology made it possible the progress in the studies of structure and expression of viral RNA genome. To manipulate the genomic RNA, it is necessary that the complementary DNA become available. Genetic modifications may be introduced in determined sites of the viral genome.
The pioneer study of David Baltimore (Racaniello, V. R., and D. Baltimore. 1981. Cloned poliovirus complementary DNA is infectious in mammalian cells. Science 214:916-9), was the first one to demonstrate that it possible to regenerate virus for the complementary DNA of the poliomyelitis virus. With the development of efficient systems in vitro transcription, it made it possible to the complete synthesis of viral RNA viral in vitro with efficiency much greater than the cDNA transcription in the cell. The development of efficient methods of cells transfection with nucleic acids, as for example electroporation and the use of cationic liposome's contributed to the increase of the transfection efficiency of cell transfection with RNA and viral regeneration. The basis of methodology of the infectious clone is established and has been used to obtain infectious clones to other virus of the positive strand.
The infectious clones may be used to better understand the molecular bases of diverse biological phenomena such as: the virulence, attenuation, mechanism of cell penetration, replication, relation with the host, conditional mutant and the design of mutants for the required functions (Bonaldo, M. C., P. S. Caufour, M. S. Freire, and R. Galler. 2000. The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines. Mem Inst Oswaldo Cruz 95 Suppl 1:215-23; Bonaldo, M. C., R. C. Garratt, P. S. Caufour, M. S. Freire, M. M. Rodrigues, R. S. Nussenzweig, and R. Galler. 2002. Surface expression of an immunodominant malaria protein B cell epitope by yellow fever virus. J Mol Biol 315:873-85).
The construction of a complete cDNA shape of the 17D vaccine virus, that can be transcript in vitro, producing RNA infectious virus, was described for the first time by Rice and colleagues (Rice, C. M., A. Grakoui, R. Galler, and T. J. Chambers. 1989. Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. New Biol 1:285-96). The virus—obtained from cDNA—was indistinguished from the parental virus, the 17D-204 subcepa, by different criteria (Rice, C. M., A. Grakoui, R. Galler, and T. J. Chambers. 1989. Transcription of infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. New Biol 1:285-96).
The acquisition of vaccines shares seeds from cDNA in good production practices was described by the first time by Marchevsky and collaborators (Marchevsky, R. S., J. Mariano, V. S. Ferreira, E. Almeida, M. J. Cerqueira, R. Carvalho, J. W. Pissurno, A. P. da Rosa, M. C. Simoes, and C. N. Santos. 1995. Phenotypic analysis of yellow fever virus derived from complementary DNA. Am J Trop Med Hyg 52:75-80), and later by Galler and Freire (patent documents U.S. Pat. No. 6,171,854 and U.S. Pat. No. 6,859,522) and Freire and collaborators (document of patent BRPI 9804283). The production process described by Freire and collaborators (patent document BRPI 9804283) may also be, in a near future, the modernization of the production of the amarilic vaccine; making it possible a significative increase in the production and improvement of the product quality (Freire, M. S., G. F. Mann, R. S. Marchevsky, A. M. Yamamura, L. F. Almeida, A. V. Jabor, J. M. Malachias, E. S. Coutinho, and R. Galler. 2005. Production of yellow fever 17DD vaccine virus in primary culture of chicken embryo fibroblasts: yields, thermo and genetic stability, attenuation and immunogenicity. Vaccine 23:2501-12).
This work created the perspective for the use of the 17D virus as an expression vector for heterolog antigens. There are several ways to obtain an expression vector from the virus with positive string RNA genome, some of which are described in published revisions by our research group (Bonaldo, M. C., P. S. Caufour, M. S. Freire, and R. Galler. 2000. The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines. Mem Inst Oswaldo Cruz 95 Suppl 1:215-23; Galler, R., M. S. Freire, A. V. Jabor, and G. F. Mann. 1997. The yellow fever 17D vaccine virus: molecular basis of viral attenuation and its use as an expression vector. Braz J Med Biol Res 30:157-68).
One of the alternatives in which our research group is working refers to the substitution of the prM/E proteins of yellow fever by the equivalent proteins of the dengue virus, so it can be obtained a chimeric virus. This approach has the advantage of the previous immunity against the vector wouldn't be a limit, since the envelope E protein contains all the epitopes for viral neutralization.
The approach of change of prM/E genes among the flavivirus was described for the first time in the patent document U.S. Pat. No. 6,184,024 and U.S. Pat. No. 6,676,936, which described the new virus with the prM/E genes of dengue 1 or 2 and the remaining of the virus genome Den 4. The first chimeric virus from 17D genome was created by change of prM/E genes of the Japanese encephalitis virus (JE) (Chambers, T. J., A. Nestorowicz, P. W. Mason, and C. M. Rice. 1999. Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties. J Virol 73:3095-101). This Chimeric was immunogenic and attenuated in monkeys, so it could promote a total protection to these animals, in face of a intracerebral challenge (IC) with the wild JE virus (Monath, T. P., I. Levenbook, K. Soike, Z. X. Zhang, M. Ratterree, K. Draper, A. D. Barrett, R. Nichols, R. Weltzin, J. Arroyo, and F. Guirakhoo. 2000. Chimeric yellow fever virus 17D-Japanese encephalitis virus vaccine: dose-response effectiveness and extended safety testing in rhesus monkeys. J Virol 74:1742-51). Recently, a clinical study in humans demonstrated that the chimerical vaccine FA/JE is safe and immunogenic in man, in similar levels to the FA 17D, with a high possibility of use, in the future, for the prevention of the Japanese encephalitis in travelers and residents in endemic regions (Monath, T. P. 2002. Japanese encephalitis vaccines: current vaccines and future prospects. Curr Top Microbiol Immunol 267:105-38; Monath, T. P., F. Guirakhoo, R. Nichols, S. Yoksan, R. Schrader, C. Murphy, P. Blum, S. Woodward, K. McCarthy, D. Mathis, C. Johnson, and P. Bedford. 2003. Chimeric live, attenuated vaccine against Japanese encephalitis (ChimeriVax-JE): phase 2 clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated Japanese encephalitis antigen. J Infect Dis 188:1213-30).
Our research group constituted four chimeric virus containing the cDNA of different dengue 2 cepas, and one of these constructions was selected for immunogenicity tests. Theses tests were performed in murine model, the results being published with the characterization of the growth and viral attenuation (Caufour, P. S., M. C. Motta, A. M. Yamamura, S. Vazquez, Ferreira, II, A. V. Jabor, M. C. Bonaldo, M. S. Freire, and R. Galler. 2001. Construction, characterization and immunogenicity of recombinant yellow fever 17D-dengue type 2 viruses. Virus Res 79:1-14).
In this strategy it was also used the creation of a chimeric virus FA 17D for the creation of a tetravalent vaccine against the different sorotypes of dengue virus (Guirakhoo, F., J. Arroyo, K. V. Pugachev, C. Miller, Z. X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratterree, and T. P. Monath. 2001. Construction, safety, and immunogenicity in nonhuman primates of a chimeric yellow fever-dengue virus tetravalent vaccine. J Virol 75:7290-304; Guirakhoo, F., K. Pugachev, J. Arroyo, C. Miller, Z. X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath. 2002. Viremia and immunogenicity in nonhuman primates of a tetravalent yellow fever-dengue chimeric vaccine: genetic reconstructions, dose adjustment, and antibody responses against wild-type dengue virus isolates. Virology 298:146-59; Guirakhoo, F., K. Pugachev, Z. Zhang, G. Myers, I. Levenbook, K. Draper, J. Lang, S. Ocran, F. Mitchell, M. Parsons, N. Brown, S. Brandler, C. Fournier, B. Barrere, F. Rizvi, A. Travassos, R. Nichols, D. Trent, and T. Monath. 2004. Safety and efficacy of chimeric yellow Fever-dengue virus tetravalent vaccine formulations in nonhuman primates. J Virol 78:4761-75, US Patent Documents U.S. Pat. No. 6,696,281 and WO0139802). In tissue culture, these chimera grow in high degrees, and were immunogenic in inoculated monks with individual formulations and tetravalent of these recombinants. But, we may stress that a higher immune response against one of the recombinant, the chimera FA/den2, due, probably, to a grater replication rate of this virus.
An ideal vaccine against the four sorotypes, as well as inducing a long-lasting response, should protect the individual against the four sorotypes efficiently, because an incomplete immunization may unleash the sickness in its more serious form. Later, other formulations were tested in monkeys, with the intention of reducing the dominant immunogenicity of the chimera FA/Den2 (Guirakhoo, F., K. Pugachev, J. Arroyo, C. Miller, Z. X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath. 2002. Viremia and immunogenicity in nonhuman primates of a tetravalent yellow fever-dengue chimeric vaccine: genetic reconstructions, dose adjustment, and antibody responses against wild-type dengue virus isolates. Virology 298:146-59). In the meantime, the adjustment of the dose for the chimera den2 resulted, in spite of a more balanced reply against the chimeric viruses types 1, 2 and 3, in a more accented reply against the chimera type 4. These results indicate that the development of a tetravalent vaccine should pass by tests with different formulations, so that an ideal adjustment may be obtained to be tested in monkeys before an optimum formulation may be attained to be used in tests of safety and immunogenicity in humans in a phase I clinical study.
The second approach refers to the insertion of the protein epitopes in the virus 17D genome of. Such insertions may be done in very immunogenic proteins of the amarilic virus, through duplication of the processing signals of the viral polyprotein by viral protease and the creation of expression cassettes—as was done with an epitope of ovalbumin, response inductor of the lymphocyte T cytotoxic, that was inserted between the genes NS2B and NS3 (McAllister, A., A. E. Arbetman, S. Mandl, C. Pena-Rossi, and R. Andino. 2000. Recombinant yellow fever viruses are effective therapeutic vaccines for treatment of murine experimental solid tumors and pulmonary metastases. J Virol 74:9197-205), Patent Documents U.S. Pat. No. 6,589,531 and US20030157128). Immunization of mice with the recombinant virus induced protection against a lethal dose of malignant melanoma cells that expressed the same epitope. It is important that the new viruses be attenuated with the vaccine 17D, that they are genetically stable and retain the immunogenic properties do heterologous antigen, promoting the correct induction of the immune response. In this sense, it should be noted that the expression of the epitope de Plasmodium yoelii through its insertion between the NS2B-NS3 genes of the virus 17D (Tao, D., G. Barba-Spaeth, U. Rai, V. Nussenzweig, C. M. Rice, and R. S. Nussenzweig. 2005. Yellow fever 17D as a vaccine vector for microbial CTL epitopes: protection in a rodent malaria model. J Exp Med 201:201-9).
It became interesting to test this system for the expression of larger genetic fragments. In this sense, our research group opted to insert the green fluorescent algae genes (GFP). This gene facilitates monitoring the infectiousness of the transcribed RNA in vitro, as from plasmidial molds, to allow the direct visualization of the synthesized proteins in transfected cultures through fluorescent microscopy.
The insertion strategy is described in FIG. 3, in which the upper part represents the genomic structure and the genetic expression. The Flavivirus genome is translated into a single polyprotein, which is cleaved by cellular proteases (↓) or viral (▾). Black vertical bars indicate transmembrane hydrophobic domains, and the asterisks indicate glycosylation sites connected to asparagine. Shadowed areas in C and prM/E represent as structural proteins present, in the mature infectious viruses. The lower part presents the general genome structure, the sequences in the cleavage sites and the proteolytic cleavages necessary for the insertion of the gene reporter between NS2A and 2B. Such strategy applies to the other sites cleaved by viral protease, situated between C-prM, NS2B-3, NS3-4A, NS4A-4B and NS4B-5.
The GFP gene was inserted between NS2A-2B and NS2B-NS3 without the recovery of the infectious virus, suggesting that the insertion of larger genetic fragments in the virus 17D genome through this approach is not possible (Bonaldo M C and Galler R, data not published).
Another manner of developing recombinant amarylic viruses having various pathogenic epitopes was the expression of protean epitopes previously classified as important in some kinds of immune replies, whether humoral or cellular, by direct insertion in the viral polyprotein. The different viral proteins contain epitopes related to the induction of the cellular reply (CTL) and humoral (formation of antibodies), in such a way that there are different possibilities of optimizing expression and immunogenicity.
A new version of the FA infectious clone was developed, containing restriction sites in the viral envelope protein gene that allowed the insertion “in-frame” of the heterologous epitopes. This was possible due to the availability of their three-dimensional structure, which allowed an analysis of the areas where insertions would be viable. A site for the insertion of the epitopes was identified in these three-dimensional analyses (f-g loop of the envelope protein), and various epitopes of different microorganisms were already inserted and expressed in the f-g loop, including epitopes de Plasmodium sp, dengue and arenavirus (Bonaldo, M. C., R. C. Garratt, M. S. Freire, and R. Galler. 2005. Novel Flavivirus vector useful for expressing heterologous antigens comprises foreign gene sequences inserted at sites in the level of its envelope protein. Great-Britain).
With relation to the Plasmodium sp epitopes, a total of 16 new viruses were created, which expressed epitopes related to the response by the T CD4+ or T CD8+ cells or the B cells. A repetitive humoral epitope of the CS surface protein of the sporozoite form of the P. falciparum was inserted in the fg loop and the virus regenerated. This virus was classified in terms of the culture growth of the cells, neutralization by soros against yellow fever and monoclonal against the epitope, this experiment proved its correct presentation in the viral surface as expected from the three-dimensional modeling, and attenuation and immunogenicity in mice (Bonaldo, M. C., R. C. Garratt, P. S. Caufour, M. S. Freire, M. M. Rodrigues, R. S. Nussenzweig, and R. Galler. 2002. Surface expression of an immunodominant malaria protein B cell epitope by yellow fever virus. J Mol Biol 315:873-85).
A recombinant Virus 17D expressing an epitope of the P. yoelii T CD8 cell, through insertion in the f-g loop, also was constructed. This virus did not have its growth in vitro characteristics altered, but showed itself more attenuated in the virulence test in mice than the virus vaccine 17DD. This epitope was correctly presented on the viral surface and is immunogenic, based on the results of immunization of mice and the Elispot tests and response with P. yoelii sporozoites, response against which was observed a protection of 70%.
Our research group also made a more detailed evaluation of the attenuation of the chimeric viruses, expressing the humoral epitopes P. falciparum and P. yoelii T CD8 through the intracerebral inoculation test in rhesus monkeys, in accordance with the requirements established by the World Health Organization for the amarilic virus vaccine. The results suggest that both the viruses are, at the minimum, as attenuated as the 17DD virus vaccine used in human vaccination. A comparative analysis of the virus envelope containing the two insertions showed that the original structural “design” of the insertion, long from the domain III involved in the connection to the receptor/tropism, was enough to not cause any alteration in the viral virulence, a fundamental aspect in the validation of this approach (Bonaldo, M. C., R. C. Garratt, R. S. Marchevsky, E. S. Coutinho, A. V. Jabor, L. F. Almeida, A. M. Yamamura, A. S. Duarte, P. J. Oliveira, J. O. Lizeu, L. A. Camacho, M. S. Freire, and R. Galler. 2005. Attenuation of recombinant yellow fever 17D viruses expressing foreign protein epitopes at the surface. J Virol 79:8602-13). This approach constitutes a recently conceded patent (Bonaldo M C, Garrat R C, Freire M S & Galler R (2001) Use of Flaviviruses for the expression of foreign protein epitopes and the development of new live attenuated vaccines for immunization against Flaviviruses and other infectious agents, GB 0105877.5 e PCT PCT/BR02/00036).
A fourth approach in the use of the 17D virus as an expression vector refers to the insertion of genes in the non translated 3′ region (NTR). This approach was done a lot in function of the variability of the length of this region in the FA virus (from Filippis, A. M., R. M. Nogueira, H. G. Schatzmayr, D. S. Tavares, A. V. Jabor, S. C. Diniz, J. C. Oliveira, E. Moreira, M. P. Miagostovich, E. V. Costa, and R. Galler. 2002. Outbreak of jaundice and hemorrhagic fever in the Southeast of Brazil in 2001: detection and molecular characterization of yellow fever virus. J Med Virol 68:620-7; Mutebi, J. P., R. C. Rijnbrand, H. Wang, K. D. Ryman, E. Wang, L. D. Fulop, R. Titball, and A. D. Barrett. 2004. Genetic relationships and evolution of genotypes of yellow fever virus and other members of the yellow fever virus group within the Flavivirus genus based on the 3′ noncoding region. J Virol 78:9652-65).
This methodology was described by Andino- and collaborators (Andino, P. R., Mcallister, M. N., 2002, Recombinant Bicistronic Flaviviruses and Methods of Use Thereof, WO 02/089840) and, basically, involved the creation of restriction sites for the insertion of expression modules. These modules, for their part, were constituted of a sequence derived from the enterovirus (Mengo or poliovirus) or from a Pest virus (Bovina Diarrhea virus), to which is directed the connection of the ribosomal sub-units in a manner that the translation of the heterologous gene may happen almost at the 3′ NTR extremity, without needing a start in the 5′ NTR region, as is characteristic of eukaryotic RNA. In this manner, the viral RNA acts as a bi-cystronic messenger, allowing the initiation of protein synthesis as from 2 RNA points, independently of the viral protein synthesis. These sequences are known as internal ribosome entry sites (IRES) Such modules vary in size, depending on the origin of the IRES and the heterologous gene to be expressed.
FIG. 4 represents the insertion of the heterologous sequences in the 3′ NTR regions of the 17D virus. The insertions of the Mengo enterovirus IRES (569 nt) and polio (663 nt) were done through cloning in restriction sites (AscI and NotI), which are adjacent to the protein P24 (693 nt) gene sequence of the human 1 immunodeficiency virus (through the NotI and Pad enzymes). The total length of the insertions varied from 1090 to 1356 nt. The restriction sites were initially introduced, as a set (AscI, NotI and PacI), exactly 25 nucleotides after the termination codon (nucleotide 10379 as from the 5′ extremity).
The transfection of the Vero in culture cells with RNA transcription in vitro, as from the cADN molds, allowed the viral regeneration referent to the constructions traced out in FIG. 3. Analysis of the resulting virus genomes, by means of nucleotide sequencing of the amplification products of this region, showed the elimination of the nucleotides. In the case of the construction with the Mengo virus IRES, the genetic instability became evident early in the first pass. The 17D-IRES-P24 virus present floating on the culture surface, presenting a cytopathic effect, had lost part of the 3′ NTR region. The termination codon remained like that as well as the first 25 nucleotides that extended up to the AscI site and more than the 22 initial IRES nucleotides. 1437 nucleotides were eliminated from this point, leaving only the last 339 nucleotides (from 508) in this region of the 17D virus. In the case of the 17D-IRES-Polio-P24 virus, the genetic instability was demonstrated by the sequencing of the 3′ NTR region of the virus present on the surface of the second pass in Vero cells. The termination codon remained intact in the genome of this virus and the first 19 nucleotides after it, following the elimination of a total of 1398 nucleotides, including the IRES and P24. The last 484 nucleotides of the original 17D virus 3′NTR region remained intact. This data showed that instability of the longer insertions in this genome region.
The genetic instability of insertions in the Flavivirus genome in the 3′ NTR region is also corroborated by the data of Pierson and collaborators (Pierson, T. C., M. S. Diamond, A. A. Ahmed, L. E. Valentine, C. W. Davis, M. A. Samuel, S. L. Hanna, B. A. Puffer, and R. W. Doms. 2005. An infectious West Nile virus that expresses a GFP reporter gene. Virology 334:28-40), to obtain the insertion of the expression modules similar to that described above, but using the GFP gene as an indicator of viral replication. Various virals isolated, analyzed after 2 passes in culture cells, led to the loss of the nucleotides that compose the IRES, as well as part do gene that codes the GFP.
The sixth possible approach in the use of the FA 17D virus for the expression of heterologous antigens refers to the development of replicons. These molecules correspond to parts of the viral genome from which the structural genes necessary for the production of viral particles were removed, although they maintained all the elements necessary for the replication of the RNA in itself. The amplification of the RNA in the transfected cells cytoplasm allows the transitory expression of heterologous genes, expression that suggests the possibility of the in vaccination (Harvey, T. J., W. J. Liu, X. J. Wang, R. Linedale, M. Jacobs, A. Davidson, T. T. Le, I. Anraku, A. Suhrbier, P. Y. Shi, and A. A. Khromykh. 2004. Tetracycline-inducible packaging cell line for production of Flavivirus replicon particles. J Virol 78:531-8; Khromykh, A. A. 2000. Replicon-based vectors of positive strand RNA viruses. Curr Opin Mol Ther 2:555-69; Tannis, L. L., A. Gauthier, C. Evelegh, R. Parsons, D. Nyholt, A. Khromykh; and J. L. Bramson. 2005. Semliki forest virus and Kunjin virus RNA replicons elicit comparable cellular immunity but distinct humoral immunity. Vaccine 23:4189-94; Westaway, E. G., J. M. Mackenzie, and A. A. Khromykh. 2003. Kunjin RNA replication and applications of Kunjin replicons. Adv Virus Res 59:99-140). Jones and collaborators (Jones, C. T., C. G. Patkar, and R. J. Kuhn. 2005. Construction and applications of yellow fever virus replicons. Virology 331:247-59) recently described a series of replicons based on the 17D virus genome. These replicons consist of the 17D virus genome deprived of the structural region that codifies the genes of the C-prM-E proteins (nucleotides 179 to 2382). Only the first 21 amino acids of C and the last 24 residues of E were kept. Three heterologous genes were inserted and expressed in the replicons in a manner dependent on the RNA replication, substituting the structural gene sequences. Meanwhile, no evidence of genetic stability of the heterologous genes, as well as studies on the immunogenicity of their products has been approached. The expression levels of the heterologous proteins also were not specified, in a way that use of this system for the development of new vaccines was not established. The principal applications of this expressions system, based on the 17D virus genome, are limited to studies on RNA viral replication mechanisms, RNA packaging and formation of viral particles.
It should be considered that the various methodologies described in this document for the insertion and expression of heterologous genes into recombinants flavivirus, as well as the object of this document, are also approaches with broad application in the expression of the whole or part of the viral genome in plasmids and DNA and RNA replicons, or even in other non-infective or infective viral systems. Khromykh, A. A., Westaway, E. G., 1997. Subgenomic replicons of the flavivirus Kunjin: construction and applications. J. Virol. 71 (2), 1497-1505; Kofler, R. M., Aberle, J. H., Aberle, S. W., Allison, S. L., Heinz, F. X., Mandl, C. W., 2004. Mimicking live flavivirus immunization with a noninfectious RNA vaccine. Proc. Natl. Acad. Sci. U.S.A. 101, 1951-1956; Aberle, J. H., Aberle, S. W., Kofler, R. M., Mandl, C. W., 2005. Humoral, and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus. J. Virol. 79, 15107-15113; Aleshin, S. E., Timofeev, A. V., Khoretonenko, M. V., Zakharova, L. G., Pashvykina, G. V., Stephenson, J. R., Shneider, A. M., Altstein, A. D. 2005. Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein. BMC Microbiology 5:45-49; Konishi, E., Kosugi, S., Imoto, J. 2006. Dengue tetravalent DNA vaccine inducing neutralizing antibody and an amnestic responses to four serotypes in mice Vaccine 24: 2200-2207; Mason, P. W., Shustov, A. V., Frolov, I. 2006). Production and characterization of vaccines based on flaviviruses defective in replication. Virology 351 432-443.
The seventh and last possible approach up to the moment, using the FA 17D virus as an expression vector, refers to the object of this current invention. In this case, given the impossibility of regenerating 17D viruses containing insertions longer than viral epitopes (>36 amino acids), whether in inter-genetic regions cleaved by viral protease or in the 3′NTR region, our group established a new approach for this purpose. This alternative is based on the insertion of the heterologous sequences—including, but not limited to those of the 10 to 2000 nucleotides—between the genes that code the E and NS1 proteins of the 17D virus. This approach is similar, theoretically, to the insertion between genes that code proteins cleaved by viral protease. Meanwhile, the cleavage between E and NS1 is done by a cellular enzyme (signalase) present in the endoplasmatic reticule, in such a manner that the cleavage sites and other structural elements necessary of viral viability are different, constituting a novelty in this methodology.
The endoplasmatic reticule serves as an entrance port for the proteins destined to all the compartments of the secreting via, that is, for the plasmatic membrane, the cell exterior and endocytic organelles. The majority of the membrane proteins and secreting via are co-traductionally integrated in the RE membrane, or pass by this to the RE lumen via specific membrane sites.
The addressing of the proteins to the RE is triggered by the presence of signal sequences in these proteins. The signal sequences are highly degenerated and essentially, uncharged, with a predominance of hydrophobic residues, and with an average size of 7 to 12 protein amino acids (von Heijne, G. 1990. The signal peptide. J Membr Biol 115:195-201).
In a first stage, the signal sequence is recognized, beginning to emerge from the tunnel exit of the ribosome during the proteic translation, by a signal recognition particle, of a ribonucleoproteic nature (SRP: “signal recognition particle); (Halic, M., and R. Beckmann. 2005. The signal recognition particle and its interactions during protein targeting. Curr Opin Struct Biol 15:116-25; Walter, P., and A. E. Johnson. 1994. Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev Cell Biol 10:87-119). Then a connection of the motif to a hydrophobic split occurs composed of a group of methionines in the SRP 54 kDa sub-unit (Keenan, R. J., D. M. Freymann, P. Walter, and R. M. Stroud. 1998. Crystal structure of the signal sequence binding subunit of the signal recognition particle. Cell 94:181-91; Lutcke, H., S. High, K. Romisch, A. J. Ashford, and B. Dobberstein. 1992. The methionine-rich domain of the 54 kDa subunit of signal recognition particle is sufficient for the interaction with signal sequences. Embo J 11:1543-51; Zopf, D., H. D. Bernstein, A. E. Johnson, and P. Walter. 1990. The methionine-rich domain of the 54 kd protein subunit of the signal recognition particle contains an RNA binding site and can be cross linked to a signal sequence. Embo J 9:4511-7). In eukaryotes, this association causes a delay in the elongation of polypeptide synthesis during the translation process. This complex connects itself to the RE membrane by a specific receptor (Keenan, R. J., D. M. Freymann, R. M. Stroud, and P. Walter. 2001. The signal recognition particle. Annu Rev Biochem 70:755-75). Both the SRP complex receptor—signal peptide and the SRP are GTPases (Egea, P. F., S. O, Shan, J. Napetschnig, D. F. Savage, P. Walter, and R. M. Stroud. 2004. Substrate twinning activates the signal recognition particle and its receptor. Nature 427:215-21; Focia, P. J., I. V. Shepotinovskaya, J. A. Seidler, and D. M. Freymann. 2004. Heterodimeric GTPase core of the SRP targeting complex. Science 303:373-7), that undergo reciprocal activation, causing the signal peptide to be released from the addressing complex and taken to the ribosome tunnel exit alignment, as to the aquatic entrance channel of the RE protein, or translocon (Beckmann, R., C. M. Spahn, N. Eswar, J. Helmers, P. A. Penczek, A. SalI, J. Frank, and G. Biobel. 2001. Architecture of the protein-conducting channel associated with the translating 80S ribosome. Cell 107:361-72; Menetret, J. F., A. Neuhof, D. G. Morgan, K. Plath, M. Radermacher, T. A. Rapoport, and C. W. Akey. 2000. The structure of ribosome-channel complexes engaged in protein translocation. Mol Cell 6:1219-32).
The translocons are comprised of various RE membrane proteins that associate themselves in such a manner as to form an aqueous pore, through which secreted proteins and domain protein lumen from the membrane pass from the cytosol to the RE (Johnson, A. E., and M. A. van Waes. 1999. The translocon: a dynamic gateway at the ER membrane. Annu Rev Cell Dev Biol 15:799-842). The translocon has an important role in the integration of the membrane proteins (Do, H., D. Falcone, 3. Lin, D. W. Andrews, and A. E. Johnson. 1996. The cotranslational integration of membrane proteins into the phospholipid bi-layer is a multi-step process. Cell 85:369-78; Heinrich, S. U., W. Mothes, J. Brunner, and T. A. Rapoport. 2000. The Sec61p complex mediates the integration of a membrane protein by allowing lipid partitioning of the transmembrane domain. Cell 102:233-44; Higy, M., T. Junne, and M. Spiess. 2004. Topogenesis of membrane proteins at the endoplasmic reticulum. Biochemistry 43:12716-22; Martoglio, B., and B. Dobberstein. 1995. Protein insertion into the membrane of the endoplasmic reticulum: the architecture of the translocation site. Cold Spring Harb Symp Quant Biol 60:41-5; Mothes, W., S. U. Heinrich, R. Graf, I. Nilsson, G. von Heijne, J. Brunner, and T. A. Rapoport. 1997. Molecular mechanism of membrane protein integration into the endoplasmic reticulum. Cell 89:523-33), therefore, in the topology of these proteins. The mechanism by which the topology of a protein is directed by the cellular translocation machinery is complex. Thus, a protein with a single membrane domain needs to translocate certain RE Lumen domains, leave others in the cytosol and guide the transmembrane segment and move the aqueous utranslocation channel to the lipidic bi-layer. Characteristics such as size and hydrophobic of the transmembrane segments, Charge distribution of the regulatory residues and size and state of the binding regulatory residues may affect the protein topology in the membrane (Beltzer, J. P., K. Fiedler, C. Fuhrer, I. Geffen, C. Handschin, H. P. Wessels, and M. Spiess. 1991. Charged residues are major determinants of the transmembrane orientation of a signal-anchor sequence. Biol Chem 266:973-8; Gafvelin, G., M. Sakaguchi, H. Andersson, and G. von Heijne. 1997. Topological rules for membrane protein assembly in eukaryotic cells. J Biol Chem 272:6119-27; Higy, M., T. Junne, and M. Spiess. 2004. Topogenesis of membrane proteins at the endoplasmic reticulum. Biochemistry 43:12716-22; Parks, G. D., and R. A. Lamb. 1991. Topology of eukaryotic type II membrane proteins: importance of N-terminal positively charged residues flanking the hydrophobic domain. Cell 64:777-87; Sakaguchi, M., R. Tomiyoshi, T. Kuroiwa, K. Mihara, and T. Omura. 1992. Functions of signal and signal-anchor sequences are determined by the balance between the hydrophobic segment and the N-terminal charge. Proc Natl Acad Sci USA 89:16-9; Spiess, M. 1995. Heads or tails—what determines the orientation of proteins in the membrane. FEBS Lett 369:76-9; von Heijne, G. 1989. Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature 341:456-8; Wahlberg, J. M., and M. Spiess. 1997. Multiple determinants direct the orientation of signal-anchor proteins: the topogenic role of the hydrophobic signal domain. J Cell Biol 137:555-62).
At the translocon entrance, the signal peptide is guided in relation to the membrane to the start of the translocation of its N- or C-terminal sequence through the membrane. The hydrophilic fraction of the polypeptide is transferred then, by the aqueous channel to the RE lumen, and the signal released laterally in the lipidic membrane. On the other side, other protein segments may stop or restart their transference to the RE or integrate themselves to the RE lipidic bi-layer as transmembrane domains (TM), and may generate proteins with multiple insertions of alpha helices in the lipidic bi-layer (Higy, M., T. Junne, and M. Spiess. 2004. Topogenesis of membrane proteins at the endoplasmic reticulum. Biochemistry 43:12716-22). The TM domains that promote integration to the membrane generally consist of 20 to 25 non polar amino acids, a size sufficient to transpass the membrane lipidic bi-layer.
FIG. 5 is referent to the processing of the Flavivirus polyprotein by cellular and viral proteases. In (A), viral polyprotein protelic sites for generation of the structural proteins, and non structural viral envelope components involved in the viral replication process. The stars (★) represent the glycosilation connected to the asparagine of certain vital proteins, the grey arrows highlight the signal peptidase cleavage sites, and the gray triangles represent the sites for the proteolysis of the viral proteolytic complex (NS2B/NS3). The (?) symbol represents the cleavage point between the NS1/NS2A viral proteins, in which acts a still undetermined cellular protease. The prM protein is later processed by the furine protease in the release of the cell viral particle (Stadler, K., Allison, S. L., Schalich, J. and Heinz, F. X. 1997. Proteolytic activation of tick-borne encephalitis virus by furin. J. Virol. 71:8475-8481). In (B), topology of the prM and E structural protein membranes, which are translocated to the cellular RE and are found associated to their membrane by means of two domains of transmembranar helices, that are indicated by cylinders. The signalase cleavage sites and the NS2B/NS3 viral protease are signed according to the nomenclature below the figure.
In Flavivirus, the polyprotein viral precursor of the structural and non structural proteins pass through the RE, membrane at various points and are processed thus: on the lumen side of the RE membrane, by the cellular enzymes, signalases, and on the cytoplasmic side, by the NS2B/NS3 proteolytic viral complex, (FIG. 5A). The RE and the viral particle assembly site, which are formed by the transport of the virions to the cell exterior, by means of the exotic or secretory via (Mackenzie, J. M., and E. G. Westaway. 2001. Assembly and maturation of the flavivirus Kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively. J Virol 75:10787-99).
Cleavage of the polyprotein in the C/prM, prM/E and E/NS1 intergenic sites, done by signalase, generate the prM and E structural proteins, that remain anchored in the luminal face of the RE membrane and form the flavivirus viral envelope. The prM and E proteins of the flavivirus envelope are type I membrane proteins (Higy, M., T. Junne, and M. Spiess. 2004. Topogenesis of membrane proteins at the endoplasmic reticulum. Biochemistry 43:12716-22; Paetzel, M., A. Karla, N. C. Strynadka, and R. E. Dalbey. 2002. Signal peptidases. Chem Rev 102:4549-80); That is, the translocation of these proteins to the RE lumen is started by the amino extremity of the polypeptide chain, which associates itself to the translocon, undergoing cleavage by signalase. This leads to the removal of the signal peptide and consequent release of the processed N-terminal from the protein to the RE lumen RE (FIG. 5B). The prM and E proteins are anchored by their carboxi-terminal in the cellular and viral membranes. These domains are composed of two hydrophobic stretches separated by a small fragment containing are least one hydrophobic residue. Thus, on the side of the RE lumen, prM and E form a stable heterodimer that will form the viral envelope (Allison, S. L., K. Stadler, C. W. Mandl, C. Kunz, and F. X. Heinz. 1995. Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J Virol 69:5816-20; Konishi, E., and P. W. Mason. 1993. Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein. J Virol 67:1672-5; Lorenz, I. C., S. L. Allison, F. X. Heinz, and A. Helenius. 2002. Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum. J Virol 76:5480-91). Thus, the prM and E viral envelope proteins have two transmembrane domains (TM1 and 2; FIG. 5, panel B), which promote their association to the lipidic bi-layer, the first, in the direction amino to the carboxi terminal of the polypeptide chain, consists of a sequence of transference stops of the protein to the RE lumen, and the second, from the signal sequence for importation and processing in the RE.
The two TM domains of the E and prM proteins form anti-parallel alpha-helices, without contact between themselves, which cross the RE Lumen membrane to the cytoplasm and Lumen again (FIG. 5, panel B). For their part, the fragment of 4 to 6 amino acids, rich in polar residues that serve as a connection between these two TM domains, appear to be associated to the internal layer of the phospholipid polar groups of the membrane (Allison, S. L., K. Stiasny, K. Stadler, C. W. Mandl, and F. X. Heinz. 1999. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. Virol 73:5605-12; Mukhopadhyay, S., R. J. Kuhn, and M. G. Rossmann. 2005. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3:13-22; Stiasny, K., S. L. Allison, A. Marchler-Bauer, C. Kunz, and F. X. Heinz. 1996. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus. J Virol 70:8142-7; Zhang, W., P. R. Chipman, J. Corver, P. R. Johnson, Y. Zhang, S. Mukhopadhyay, T. S. Baker, J. H. Strauss, M. G. Rossmann, and R. J. Kuhn. 2003. Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol 10:907-12).
The protein of capsid (C) is separated from the prM, precursor protein of the membrane protein or M, by a signal sequence that directs the translation of the prM. Meanwhile, so that cleavage of the peptide signal occurs and formation of the COOH terminal of the C protein C and the prM N-terminal, it is strictly necessary that the NS2B/NS3 proteolytic complex first catalyzes the COOH terminal COOH of the C protein on the cytoplasmatic side of the RE membrane RE (FIG. 5B). This is the only site of the polyprotein region containing the structural proteins that are processed by this enzyme (Amberg, S. M., A. Nestorowicz, D. W. McCourt, and C. M. Rice. 1994. NS2B-3 proteinase-mediated processing in the yellow fever virus structural region: in vitro and in vivo studies. J Virol 68:3794-802; Lobigs, M. 1993. Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3. Proc Natl Acad Sci USA 90:6218-22; Yamshchikov, V. F., and R. W. Compans. 1993. Regulation of the late events in flavivirus protein processing and maturation. Virology 192:38-51). It is only after this cleavage that the cleavage of the signal peptide by the signal peptidase happens, probably due to the conversion of the cleavage signal peptidase site from a cryptic conformation to an accessible one (Lobigs, M. 1993. Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3. Proc Natl Acad Sci USA 90:6218-22). The cleavage process of the prM protein signal peptide by the signal peptidase is modulated by the initial hydrolysis of the C protein C-terminal by viral protease. Thus, it is only after the cleavage and generation of the mature C protein that the hydrolysis of the signal peptide occurs, and the consequent release of the prM protein N-terminal in the RE lumen. This stage is preserved between the Flavivirus, indicating its regulatory nature during the processing of the polyprotein structural region (Amberg, S. M., and C. M. Rice. 1999. Mutagenesis of the NS2B-NS3-mediated cleavage site in the Flavivirus capsid protein demonstrates a requirement for coordinated processing. J Virol 73:8083-94; Stocks, C. E., and M. Lobigs. 1998. Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM. J Virol 72:2141-9). In this sense, it was shown that this coordinated processing is critical for the incorporation of the nucleocapsid during the formation of the viral particles in the RE (Lee, E., C. E. Stocks, S. M. Amberg, C. M. Rice, and M. Lobigs. 2000. Mutagenesis of the signal sequence of yellow fever virus prM protein: enhancement of signalase cleavage In vitro is lethal for virus production. J Virol 74:24-32; Lobigs, M., and E. Lee. 2004. Inefficient signalase cleavage promotes efficient nucleocapsid incorporation into budding flavivirus membranes. J Virol 78:178-86; Stocks, C. E., and M. Lobigs. 1998. Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM. J Virol 72:2141-9). Therefore, for coordination of the cytosolic cleavages, and the RE lumen RE in the C/prM junction, it is indispensable that an efficient incorporation of the nucleocapsid to the membranes containing the viral envelope proteins occurs, because the brewing of the subviral particles, containing only the viral envelope proteins, do not depend on the C protein or the assembly of the nucleocapsid (Allison, S. L., K. Stadler, C. W. Mandl, C. Kunz, and F. X. Heinz. 1995. Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J Virol 69:5816-20; Lorenz, I. C., S. L. Allison, F. X. Heinz, and A. Helenius. 2002. Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum. J Virol 76:5480-91).
The C-terminal portion of the prM protein contains two adjacent hydrophobic stretches, interrupted by a charged residue; that act, the first transmembrane stretch, as a stop signal for the prM transference, and the second, as a signal sequence for the translocation of the E protein to the RE (Markoff, L. 1989. In vitro processing of dengue virus structural proteins: cleavage of the pre-membrane protein. J Virol 63:3345-52; Ruiz-Linares, A., A. Cahour, P. Despres, M. Girard, and M. Bouloy. 1989. Processing of yellow fever virus polyprotein: role of cellular proteases in maturation of the structural proteins. J Virol 63:4199-209). Two adjacent transmembrane sequences act in the same manner, through the stoppage of the E protein translocation and the entrance of the RE from the NS1 protein. In a general fashion, the processing by signal peptidases is important for the importation of the prM, E and NS1 proteins to the RE, and for the generation of their extreme N-terminal.
Cocquerel and collaborators (Cocquerel, L., C. Wychowski; F. Minner, F. Penin, and J. Dubuisson. 2000. Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, sub-cellular localization, and assembly of these envelope proteins. J Virol 74:3623-33), when they analyzed the C-terminal sequences of the Flavivirus viral envelope proteins, could demonstrate that this organization is very similar to that found in the Hepatitis C virus and in other members of the Flaviviridae Family. It can also be determined, that the sequences which connect the two TM domains, within the different groups, have specific standards related to these different virus groups; but the presence of at least one positively charged group (R or K) in this region was general, indicating an important function. The comparison of this fragment between different virus groups of the Flaviviridae family point to a wide variability of the amino acid sequences of the connection segment of the TM domains TM between these different groups, indicating that these should be related to molecular interactions that would occur specifically within these groups (Cocquerel, L., C. Wychowski, F. Minner, F. Penin, and J. Dubuisson. 2000. Charged residues in the transmembrane domains of hepatitis C virus glycoproteins play a major role in the processing, sub-cellular localization, and assembly of these envelope proteins. J Virol 74:3623-33). Notably, the connection segments of the TM segments of the structural proteins in Flavivirus are longer than their counterparts in other groups, presenting various polar residues preserved (N, Q, S and/or T). Another characteristic consists of the fact that the second Flavivirus TM domain is noticeably larger, with around 19 residues, in relation to the other viral groups of the family, with around 12 to 13 residues. Mutations in the prM and E TM domains affect the formation of the subviral particles or effective viruses, but appear not to affect the heterodimerization capacity of the prM and E proteins, indicating that these domains are sensitive to a change in their amino acid sequence, and the interactions between the alpha helices of the domains have a role in the formation of the viral envelope (Op De Beeck, A., R. Molenkamp, M. Caron, A. Ben Younes, P. Bredenbeek, and J. Dubuisson. 2003. Role of the transmembrane domains of prM and E proteins in the formation of yellow fever virus envelope. J Virol 77:813-20). Recently, it could be established that the chimeric proteins, expressing these Flavivirus prM and E protein transmembrane domains, situated themselves mainly in the RE, indicating that these domains contain retention signals in the RE. It is probable that accumulation of these proteins in the RE occurs, leading to the heterodimerization of these and the brewing of the immature viral particles in the RE lumen, as from which will start the secretion via of the virions to the extra-cellular medium.
In relation to the Flavivirus E protein, these TM domains make part of other structural elements situated in the last one hundred amino acid residues of the C-terminal of this protein, a region denominated stem-anchor (Allison, S. L., K. Stiasny, K. Stadler, C. W. Mandl, and F. X. Heinz. 1999. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. J Virol 73:5605-12). This region is not part of the three-dimensional structure elucidated for the E protein ectodomain of different Flaviviruses, due to its hydrophobic character (Modis, Y., S. Ogata, D. Clements, and S. C. Harrison. 2003. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc Natl Acad Sci USA 100:6986-91; Rey, F. A., F. X. Heinz, C. Mandl, C. Kunz, and S. C. Harrison. 1995. The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 375:291-8). In the TBE virus E protein, the stem-anchor region covers the residues from 401 to 496 (Allison, S. L., K. Stiasny, K. Stadler, C. W. Mandl, and F. X. Heinz. 1999. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. J Virol 73:5605-12; Stiasny, K., S. L. Allison, A. Marchler-Bauer, C. Kunz, and F. X. Heinz. 1996. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus. J Virol 70:8142-7)
The stem region connects the E protein ectodomain with the transmembrane region. This domain is composed of two alpha-helices, denominated H1 and H2, separated by a connection sequence (CS) highly preserved in the Flavivirus, see FIG. 7A (Stiasny, K., Allison, S. L., Marchler-Bauer, A., Kunz, C. and F. X. Heinz. 1996. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus. J. Virol. 70: 8142-8147; Allison, S. L., Stiasny, K., Stadler, K., Mandl, C. W. and F. X. Heinz. 1999. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. J. Virol. 73, 5605-5612). The first helix, H1, forms an angle with the external layer of membrane lipids and the second, H2 finds itself placed above the side of the external membrane, with the hydrophobic side turned to the hydrophobic side of the membrane (Mukhopadhyay, S., R. J. Kuhn, and M. G. Rossmann. 2005. A structural perspective of the flavivirus life cycle. Nat Rev Microbiol 3:13-22; Zhang, W., P. R. Chipman, J. Corver, P. R. Johnson, Y. Zhang, S. Mukhopadhyay, T. S. Baker, J. H. Strauss, M. G. Rossmann, and R. J. Kuhn. 2003. Visualization of membrane protein domains by cryo-electron microscopy of dengue virus. Nat Struct Biol 10:907-12). It is postulated that the stem region makes contact with the side of the E protein closest to the lipidic membrane, neutralizing the electrostatic repulsion between the phospholipid radicals of the external lipidic membrane and the interior surface of the E protein ectodomain (Zhang, Y., W. Zhang, S. Ogata, D. Clements, J. H. Strauss, T. S. Baker, R. J. Kuhn, and M. G. Rossmann. 2004. Conformational changes of the flavivirus E glycoprotein. Structure (Camb) 12:1607-18). The H1 region appears to be involved in the formation of E protein homotrimers during the fusion process (Allison, S. L., K. Stiasny, K. Stadler, C. W. Mandl, and F. X. Heinz. 1999. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. J Virol 73:5605-12). In this way, truncated proteins lacking the stem-anchor domains are secreted as dimers, undergo dissociation in acid pH, which causes the fusion process, but does not manage to form trimers. On the other side, proteins truncated immediately after H1 may form trimers in low pH, indicating that this region may be involved in the conversion of monomers to trimers during the fusion process to the endosomic membrane. The second stem element, CS, is highly preserved in Flavivirus (Stiasny, K., S. L. Allison, A. Marchler-Bauer, C. Kunz, and F. X. Heinz. 1996. Structural requirements for low-pH-induced rearrangements in the envelope glycoprotein of tick-borne encephalitis virus. J Virol 70:8142-7), indicating a still undefined important function.
The second amphipatic element of the stem—H2, jointly with the first transmembrane domain (TM1), are important for the stability of the prM/E dimer and may be interacting directly with prM.
As was previously discussed, the two TM1 and TM2 transmembrane elements of the E protein C-terminal constitute a membrane double anchor. The TM2 domain appears to be dispensable in the formation of subviral particles (Allison, S. L., K. Stiasny, K. Stadler, C. W. Mandl, and F. X. Heinz. 1999. Mapping of functional elements in the stem-anchor region of tick-borne encephalitis virus envelope protein E. J Virol 73:5605-12), meanwhile it is an important functional component in the formation of viral particles and viral infection, because it functions as a signal peptide for the translocation of the NS1 protein to the RE lumen.